A system that delivers an antioxidant to mitochondria for the treatment of drug-induced liver injury

Mitochondria, a major source of reactive oxygen species (ROS), are intimately involved in the response to oxidative stress in the body. The production of excessive ROS affects the balance between oxidative responses and antioxidant defense mechanisms thus perturbing mitochondrial function eventually leading to tissue injury. Therefore, antioxidant therapies that target mitochondria can be used to treat such diseases and improve general health. This study reports on an attempt to establish a system for delivering an antioxidant molecule coenzyme Q10 (CoQ10) to mitochondria and the validation of its therapeutic efficacy in a model of acetaminophen (APAP) liver injury caused by oxidative stress in mitochondria. A CoQ10-MITO-Porter, a mitochondrial targeting lipid nanoparticle (LNP) containing encapsulated CoQ10, was prepared using a microfluidic device. It was essential to include polyethylene glycol (PEG) in the lipid composition of this LNP to ensure stability of the CoQ10, since it is relatively insoluble in water. Based on transmission electron microscope (TEM) observations and small angle X-ray scattering (SAXS) measurements, the CoQ10-MITO-Porter was estimated to be a 50 nm spherical particle without a regular layer structure. The use of the CoQ10-MITO-Porter improved liver function and reduced tissue injury, suggesting that it exerted a therapeutic effect on APAP liver injury.

www.nature.com/scientificreports/ devices into nanocarriers such as a poly-lactic-co-glycolic acid (PLGA) copolymer and solid dispersions are often used 14,15 . In contrast, little information is currently available on the use of phospholipids as a major component of LNPs. Encapsulating CoQ 10 as a model drug into an LNP by a microfluidic device was reported to result in an emulsion-based solid dispersion 16 , but its application to LNPs has not been extensively investigated. We previously reported on a method for preparing a CoQ 10 -MITO-Porter, a mitochondria-targeted LNP encapsulating CoQ 10 , using a microfluidic device. The procedure had a high degree of reproducibility and could be scaled up 17 . However, the lipid composition and internal structure of the CoQ 10 -MITO-Porter was not discussed and it is important to confirm whether or not this formulation can function in vivo. In general, the internal structure of LNPs is characterized by the structure and polarity of the lipid molecules, the lipid composition ratio, and ratio of encapsulated molecules to lipids. The internal structure of the LNP may be affected by the encapsulated molecules. The present study reports on attempts to confirm that polyethylene glycol (PEG), octaarginine (R8), a functional ligand, and CoQ 10 can be incorporated into an LNP preparation using a microfluidic device and to structurally characterize the resulting CoQ 10 -MITO-Porter by Transmission electron microscope (TEM) observations and small angle X-ray scattering (SAXS) measurements. To achieve this, a mouse model of an acetaminophen (APAP) liver injury was created to evaluate the therapeutic effect of the CoQ 10 -MITO-Porter (Fig. 1). APAP liver injury is a type of mitochondrial-related disease caused by increased oxidative stress 18,19 . The delivery of antioxidant molecules to mitochondria, the major source of ROS, has the potential to be an effective therapeutic strategy for the treatment of this type of disease. The CoQ 10 -MITO-Porter would be expected to normalize ROS levels and mitochondrial function against APAP liver injury. The biodistribution of the CoQ 10 -MITO-Porter was followed, and its therapeutic effect was evaluated using a blood biochemical test and histopathological observations.
Preparing the LNP using a microfluidic device required the inclusion of PEG or R8 in the lipid composition to ensure particle dispersion. We therefore concluded that PEG is essential for preparing LNPs that contain poorly water-soluble molecules such as CoQ 10 .
Evaluation of the internal structure of CoQ 10 -MITO-Porter particles. The structure of the CoQ 10 -MITO-Porter was evaluated after being prepared by a microfluidic device and after dialysis. TEM observations showed that the CoQ 10 -MITO-Porter is a spherical particle with a diameter of approximately 50 nm (Fig. 4A). SAXS measurements were employed to characterize the fine internal structure of the particles (Fig. 4B). In general, the scattering intensity profiles obtained from this measurement method are useful for predicting the size distribution, shape and structure of particle samples. The CoQ 10 -MITO-Porter and the empty-MITO-Porter were used to compare whether encapsulating CoQ 10 or not makes a difference to the morphology of the LNP. Broad peaks appeared at 0.8 nm −1 for CoQ 10 -MITO-Porter (blue profile in Fig. 4B) and at 0.9 nm −1 for the empty-MITO-Porter (red profile in Fig. 4B). In SAXS measurements, nanoparticles with a lamellar structure and nanoparticles without a periodic structure show a sharp peak and a broad peak, respectively 20,21 . The CoQ 10 -MITO-Porter and the empty-MITO-Porter were inferred to be tight structures with no lamellar phase based on the appearance of broad peaks. These results were consistent with the TEM observations. The drug/ lipid (w/w) of CoQ 10 -MITO-Porter is 0.19 ± 0.03 17 . The fine structure of the MITO-Porter was not changed by the presence of CoQ 10 . The position of the peak reflects the particle size. The smaller the particle, the more the peak shifts towards the wide-angle side 22 . The peak for the empty-MITO-Porter was shifted towards the wideangle side compared to the CoQ 10 -MITO-Porter, suggesting a smaller diameter (Table S5). This is also consistent with our evaluation of the physical properties of the LNPs.
Based on the results of TEM observations and SAXS measurements, a conceivable image of fine structure of the CoQ 10 -MITO-Porter was shown in Fig. 4C. These results suggest that the CoQ 10 -MITO-Porter is a spherical particle with a non-lamellar interior form of approximately 50 nm. The fine structure of the LNPs would be expected to ensure that the molecule functions as a drug, because of the stability and the release of the formulation.
Tissue migration of CoQ 10 -MITO-Porter. The biodistribution of the CoQ 10 -MITO-Porter was evaluated by ex vivo imaging systems in normal and APAP liver injury model mice (Fig. 5). The amount transferred and rate of transfer into each tissue were calculated based on the observed images. Several major tissues were observed at 3 h after the intravenous administration of the 1,1'-dioctadecyl-3,3,3' ,3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD)-labelled CoQ 10 -MITO-Porter (Fig. 5C). In the case of the APAP liver injury model mice, the CoQ 10 -MITO-Porter was administrated at 1 h after the APAP treatment. The CoQ 10 -MITO-Porter accumulated at the highest levels in the liver among the major tissues. The accumulation of the LNPs in the APAP-induced liver injury group was significantly increased compared to the non-treated group (Fig. 5A). The CoQ 10 -MITO-Porter was delivered to the liver, followed by the spleen, lung, kidney, and heart. The APAP-treated mice tended to lose the CoQ 10 -MITO-Porter from the blood. No differences between the two groups were found in the transfer rate into each tissue. The transfer rate of the LNPs to the liver was approximately 40%. Table 2. Two-way ANOVA analysis results of turbidity of the MITO-Porter solution. Absorbance intensity of the MITO-Porters are performed a 2-way ANOVA analysis to compare the effect of 2 factors that are "PEG" and "R8". A significant interaction between the two factors was found and then a simple main effect test performed. p < 0.001 by simple main effect test, followed by Tukey test. www.nature.com/scientificreports/ Accordingly, the CoQ 10 -MITO-Porter appears to be transferred to the liver by intravenous administration, meaning that it could be applied to the treatment of liver diseases such as an APAP liver injury.
Evaluation of the therapeutic effect of the CoQ 10 -MITO-Porter against the APAP liver injury model. The therapeutic effect of the CoQ 10 -MITO-Porter was tested against an APAP-induced liver injury ( Fig. 6). APAP is oxidized by CYP2E1 to the active metabolite N-acetyl-p-benzoquinone imine (NAPQI) in the liver. NAPQI increases the oxidative stress associated with mitochondrial dysfunction, developing liver damage. Since CYP2E1 is a cytochrome P450 molecule, a drug-metabolizing enzyme in every animal species, APAP liver injury can be reproduced by a single dose in experimental animals, which cause acute toxicity symptoms that are very similar to that in humans. In the case, the drug-induced liver injury model was created as a system for evaluating drug-induced liver injury, and was used to clarify the mechanisms of hepatotoxicity and to evaluate the therapeutic effect of the preparation. Mice with APAP hepatotoxicity can be produced with mild symptoms: serum alanine aminotransferase (ALT) levels < 1000 IU/L and moderate models: serum ALT levels ~ 3000 IU/L according to the feeding conditions and the drug loading dose, they were often assessed at an early stage: 6-8 h, an intermediate stage: 12 h and a late stage: 24 h after the APAP treatment [23][24][25][26] . In the present experiment, 24 h after injection was selected as the evaluation time to evaluate the persistence of the protective effect of   www.nature.com/scientificreports/ trend to that for serum ALT (Fig. S3). In a preliminary study, the empty-MITO-Porter was, as expected, found to have no hepatoprotective effect (Fig. S4). The therapeutic effect was assessed by liver histology using hematoxylin and eosin (H&E) stains and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) stains, the degree of tissue damage and the presence of apoptosis, respectively (Fig. 6D). In HE-stained sections of the PBS (−) group and the CoQ 10 suspension group, a necrotic area extended around the portal vein (blue dotted line in Fig. 6D) and morphological abnormalities such as loss or shrinkage of the nucleus and cell swelling were observed. On the contrary, few necrotic images were observed in the CoQ 10 -MITO-Portert group and hepatocyte  www.nature.com/scientificreports/  Figure S3 showed serum LDH. Figure S4   www.nature.com/scientificreports/ sequence regularity was maintained. Percentage of necrotic area was quantitatively determined based on HEstained images (Fig. 6C). The quantitative results showed significant differences between the CoQ 10 -MITO-Porter group and the other groups. In TUNEL-stained sections of the PBS (−) and CoQ 10 suspension groups, brown apoptosis-positive regions were observed in extensive areas of the liver, indicating that cell death was induced by oxidative stress caused by APAP hepatotoxicity. A few brown areas were observed in the CoQ 10 -MITO-Portert group. Thus, the histological evaluation also showed that CoQ 10 -MITO-Porter reduced APAP liver injury. The results from these animal experiments indicate that administration of CoQ 10 -MITO-Porter is effective in APAP liver injury by delivering the antioxidant CoQ 10 to the liver, thereby reducing oxidative stress and restoring tissue damage and biochemical functions.

Discussion
APAP liver injury is triggered by mitochondria-induced oxidative stress. The application of antioxidant molecules to APAP-damaged liver injury is considered to be an effective preventive and therapeutic strategy. Previous studies have attempted to use CoQ 10 , resveratrol and 2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl) triphenylphosphonium chloride (Mito-TEMPO). For example, in an experimental system in which APAP liver injury model mice (C57BL/6 J, male, 8 weeks old, fasting) were administered CoQ 10 solution (5 mg/kg CoQ 10 in a soybean lecithin solution, intraperitoneal administration) at 1.5 h after APAP administration, the serum ALT levels in the PBS (−) group were approximately 2400 IU/L, whereas that in the CoQ 10 group they were approximately 1500 IU/L, suggesting improved liver function 24 . The CoQ 10 -MITO-Porter (0.9 mg/kg CoQ 10 , intravenously administered at 1 h post-APAP injection) showed significantly decreased serum ALT levels, indicating that it suppressed liver injury (Fig. 4B). The CoQ 10 -MITO-Porter produced a therapeutic effect at CoQ 10 concentrations as low as 0.9 mg/kg. We therefore conclude that, based on the therapeutic evaluation, that the particle designs of the LNPs, which contained soluble forms of CoQ 10 and the regulation of the CoQ 10 pharmacokinetics, allowed the particles to be transferred to liver mitochondria following its introduction into hepatocytes in the liver. The use of microfluidic devices as a method for preparation also largely contributed to the formation of stable LNPs as small as 50 nm, which was difficult using the batch method. The purpose of the present study was to examine the therapeutic effects of CoQ 10 delivery to mitochondria to alleviate oxidative stress levels by eliminating mitochondrial dysfunction. The major differences between the findings reported in the previous study and the current study are the size of the LNPs and the dose of CoQ 10 . In general, it is more difficult to prove that a treatment is effective than to prove a prevention effect. A previous study demonstrated the hepatoprotective effect of the preventive administration of a conventional 80 nm CoQ 10 -MITO-Porter (2 mg/kg CoQ 10 ) in a mouse model of hepatic ischemia-reperfusion 27 . The formulary difference in the novel CoQ 10 -MITO-Porter was that low CoQ 10 dosages could provide significant therapeutic effects with low burdens to the body.
In the first step in the preparation of LNPs an invasive lipid nanoparticle production (iLiNP®) device, which contains a flow channel structure (10 sets of baffle mixer structures) and flow conditions (Total flow = 500 µL/ min, Flow rate ratio = 4) in the microfluidic device, enable the rapid mixing of lipids/ethanol solutions and buffer solutions, thus leading to the formation of small-sized nanoparticles 28 . Specifically, this led to a shorter growth process by fusion and aggregation of the lipid-associated complex, namely, the lipid core, the lipid disc and LNP formation. Secondly, during the dialysis process, in solutions containing 20% ethanol, the LNPs tended to restructure, maintaining a thermodynamically stable dispersion in aqueous solution according to their lipid properties and composition. The MITO-Porter [PEG (−), R8 (−)], composed of neutral lipids DOPE/SM, self-assembles through intermolecular hydrophobic interactions. However, the thermodynamically unstable particles formed aggregates under the present preparation conditions using a microfluidic device, that produces a rapid polarity change with dilution of the ethanol solution, and the dialysis process completely replaces the aqueous solvent. As a result, CoQ 10 was precipitated out when it reached a supersaturated state (Figs. 1, 2, S1, S2).
The charged PEG and R8 are oriented on the LNP surface, creating an electrostatic repulsive force that inhibits fusion and aggregation between particles. The MITO-Porter [PEG (+), R8 (+)] possessed a zeta potential of approximately 10 mV that was derived from R8 (10 mol% of total lipids), that causes the LNPs to be stabilized by electrostatic repulsive forces. PEG is located on the outer layer of the LNP, exerting a protective colloidal action to form a hydration layer and a size-controlling effect 29 . In the CoQ 10 -MITO-Porter [PEG (+), R8 (+)] caused the formation of stable particles due to the effect of PEG. On the other hand, the empty-MITO-Porter [PEG (+), R8 (+)] could also be prepared, but its PDI was higher than that of the CoQ 10 -MITO-Porter. After a microfluidic device preparation, the particle size was very small, 34.2 ± 6.7 nm (PDI = 0.257 ± 0.0187) (Fig. S1, Table S1). Thermodynamically unstable nanoparticles tended to aggregate because, in small-sized nanoparticles with a high curvature there was a substantial distance between the lipid molecules, allowing easy contact between hydrophobic regions of lipids and water molecules during the dialysis. The PEG and R8 ratios in the present study are the optimized conditions for the preparation of CoQ 10 -MITO-Porter. An Empty-MITO-Porter should be optimized to ensure dispersion stability.
Preparing LNPs requires the inclusion of materials in the final product that inhibit the excessive self-assembly of lipids and provide for the formation of a stable dispersion system. PEG was found to be a particularly important factor in enhancing dispersion stability and to efficiently package for poorly water-solubility molecules such as CoQ 10 . PEG can also control pharmacokinetics including blood retention and physicochemical properties of the formulation by differences in the head structure and lipid scaffold structure 30 . To achieve purposeful LNP preparation an adequate PEG should be used and the ratios should be optimized.
Biodistribution evaluations showed that CoQ 10 -MITO-Porter largely accumulated in the liver after intravenous administration. In the case of induced liver injury, the delivery of LNPs to the liver was increased significantly (Fig. 5). CoQ 10 -MITO-Porter without R8 modification significantly decreased accumulation in liver and liver mitochondria 27 . The CoQ 10 -MITO-Porter was modified with R8 on the particle surface. The data for the www.nature.com/scientificreports/ resulting CoQ 10 -MITO-Porter were in agreement with previous reports in that R8-modified nanoparticles tend to accumulate in the liver 31 . The delivery of therapeutic molecules to hepatocytes is an important therapeutic strategy for the treatment of an APAP liver injury in order to reduce the levels of NAPQI, a toxic metabolite of APAP, in hepatocytes 18,19 . The capillary walls of the liver contain numerous small pores called fenestra. Therefore, the LNPs must pass through the fenestra and eventually reach hepatocytes. Since the fenestra diameter averages 141 nm and 107 nm for mice and humans, respectively 32 , the particle size needs to be controlled to less than 100 nm. The administration of 60 nm nanoparticles to mice caused a reduced APAP hepatotoxicity by allowing them to pass through the fenestra and easily access hepatocytes, as has been previously reported 33 . From our previous studies, an 84 nm CoQ 10 -MITO-Porter has been observed in hepatocytes when administered intravenously to mice 27 . The 50 nm CoQ 10 -MITO-Porter used in this study was also expected to be transferred to hepatocytes similar to the previous study. In APAP liver injury, sinusoidal endothelial cells are damaged before hepatocytes, leading to the disruption of the sinusoidal wall and the mobilization of leukocytes to the inflammation sites 34 . The nanoparticles that were modified with R8 were opsonized in the liver and recognized by macrophages 29 . In Fig. 5, mice with an APAP liver injury model likely have increased LNP migration compared to normal mice because of the enlargement of fenestra and the increased mobilization of hepatic macrophages. Moreover, it will be necessary to check whether CoQ 10 is delivered to mitochondria by tracking the particle dynamics in the future. Lipid-based drug delivery contributes to the achievement of therapeutic strategies by the selection of ligands with an affinity for specific target tissues, cells, and cell organelles and the control of particle size. The use of the CoQ 10 -MITO-Porter in the APAP liver injury model resulted in reducing oxidative stress, namely, improved ALT levels, decreased areas of tissue necrosis, and suppressed apoptosis (Fig. 6). Serum ALT levels were correlated with the histologic evaluation. The CoQ 10 -MITO-Porter group exhibited significantly lower percentage of necrotic area than the PBS (−) group and the CoQ 10 suspension group (p < 0.01). Although NAPQI is produced at optimal doses, the detoxification mechanism functions to inactivate it by using GSH. An APAP overdose in mice causes the depletion of GSH within 30 min, after which, the NAPQI combines with intracellular proteins, and the maximum amount of NAPQI-protein complex is reached within 30 min to 1 h 23 . NAPQI binds to mitochondrial proteins and induces mitochondrial dysfunction and destruction due to increased oxidative stress, which then results in the release of apoptosis-inducing factors (AIF), such as endonuclease G and cytochrome c from mitochondria, followed by the initiation of DNA damage and necrotic cell death 18,19 . These phenomena were clearly observed in both the PBS (−) group and CoQ 10 suspension group. For this reason, the administration of CoQ 10 -MITO-Porter 1 h after APAP loading constitutes therapeutic treatment.
Regarding therapeutic targeting organelles for APAP liver injury, mitochondria are attractive because they play central roles in the signaling of numerous important cellular events 19 . ROS are primarily produced by the electron transfer system that is located in the mitochondrial inner membrane 18 . It would therefore by expected that delivering antioxidant molecules such as CoQ 10 to the interior of mitochondria would effectively scavenge excessive ROS caused by oxidative stress. It was previously shown that a radio isotope-labeled CoQ 10 -MITO-Porter accumulates in liver mitochondria 27 . In a previous study, hepatoprotective effects were compared between LNPs with different mitochondrial fusion activity in a mouse model of hepatic ischemiareperfusion 27 . The CoQ 10 -MITO-Porter improved ALT levels, but the CoQ 10 -loaded LNP (CoQ 10 -EPC-LP) used in that study was composed of mitochondrial low fusion lipids, showed higher levels. In the evaluation of mitochondrial fusion activity using these two LNPs, the CoQ 10 -MITO-Porter showed fusion activity, while CoQ 10 -EPC-LP had little fusion ability. It is possible that the CoQ 10 -MITO-Porter in the present study has mitochondrial fusion properties because it has the same lipid composition as the LNPs used in the previous study. CoQ 10 functions as a coenzyme for ATP production in the mitochondrial electron transfer system, and would be expected to improve mitochondrial function. The mechanism responsible for the therapeutic effect was not fully elucidated in this study. Additional studies will be needed to evaluate oxidative stress markers and mitochondrial function. The delivery of CoQ 10 to mitochondria by the MITO-Porter system is one of the important therapeutic strategies against mitochondria-related diseases such as APAP liver injury that is associated with oxidative stress.
Experimental animals. C57BL/6 J male mice (8-10 weeks old, ~ 21 g) were purchased from Hokudo Co., Ltd (Sapporo, Japan). The mice were housed at a 12 h light-dark cycle with free intake of water and standard mouse food in a specifically pathogen-free room, acclimated to the breeding environment for a week and then received each treatment. From an animal welfare concept, it was decided to evaluate the small number of cases for which a statistical analysis could be performed. Euthanasia was performed by cervical dislocation while isoflurane inhalation anesthesia was used in cases of sufficient activity. All animal protocols were approved by the institutional Animal Care and Research Advisory Committee of the Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan (date: March 17, 2022, registration no. 18-0096).
Preparation of the CoQ 10 -MITO-Porter. The CoQ 10 -MITO-Porter was prepared by a microfluidics system using a iLiNP® device (basic structure of 10 baffle mixer structure sets, standard dimensions of the baffle mixer structure: a width of 150 μm, a depth of 100 μm and an interval of 100 μm). 4 Biodistribution of the CoQ 10 -MITO-Porter. The mice were fasted for 21 ± 3 h pre first injection and allowed only drinking water. The non-treated and APAP-treated groups received PBS (−): 10 µL/g and APAP solution: 200 mg/10 mL/kg intraperitoneally, respectively, and 1 h later, both groups were injected intravenously with DiD-labeled CoQ 10 -MITO-Porter at a lipid dosage of 20 nmol/8 µL/g. Three hours after LNP administration, the mice were collected, whole blood was obtained by cardiac blood sampling and sacrificed. Subsequently, the heart, lung, liver, spleen, and kidneys were harvested immediately, washed with PBS (−). The organs were imaged using a FluorVivo™ 300 (INDEC BioSystems, Los Altos, CA, USA). DiD was excited with a 644 nm light, and the red filter of the fluorescence detection channel was set for DiD. All images were analyzed using Image J software (http:// rsb. info. nih. gov/ ij/). The transfer rate on several main organs was calculated as follows: quantified fluorescence efficiency of an objective organ/ quantified fluorescence efficiency of all organs × 100.
Therapeutic effect validation against APAP liver injury model mice. For determining the therapeutic effects of CoQ 10 -MITO-Porter, mice were randomly divided into 3 groups of 6, 3 and 3 mice, named PBS (−), CoQ 10 suspension and CoQ 10 -MITO-Porter groups, respectively. APAP was dissolved in PBS (−), heated at 60 °C for 30 min. The CoQ 10 suspension was prepared by dispersing in PBS (−), sonicated and heating at 50 °C for 5 min. Samples containing CoQ 10 were administered to mice at a dose of 0.9 mg/kg CoQ 10 in a total volume of 8 mL/kg. The fasted mice were injected intraperitoneally with 200 mg/kg APAP. After 1 h, The PBS (−) and CoQ 10 -MITO-Porter groups were administered intravenously and the CoQ 10 suspension group intraperitoneally. At 6 h after APAP treatment, blood was collected from the mice, followed by euthanasia. To measure serum ALT levels, a marker of liver function, blood was incubated at room temperature for 1 h and serum was obtained by centrifugation (800g, 4 °C, 5 min). Serum ALT value was determined by Pure Auto S ALT-L kit (Sekisui Medical Co., Ltd, Tokyo, Japan). For the evaluation of the areas of necrosis and DNA fragmentation, the liver was harvested immediately after euthanasia, washed with PBS (−), and stored in 4% (v/v) paraformaldehyde in PBS (−) for 48 h at 4 °C. Paraffin-embedded liver section, HE staining and TUNEL staining were performed by Morphotechnology Co., Ltd (Sapporo, Japan). Stained liver sections were observed with the SMZ 1270i (Nikon Corporation, Tokyo, Japan). HE-stained sections were analyzed to identify percentage of necrotic areas using Image-pro Plus 7.0. The percentage of necrosis was calculated as the ratio of necrotic areas to whole tissue areas.

Statistical analysis.
The data shown in Figs. 2, 3A, 5A,B, 6B,C are expressed as the mean ± standard deviation (S.D.) for the indicated number of experiments. In Figs. 2 and 3A, we performed 2-way ANOVA, followed by Tukey test to compare the effect of 2 factors. If a significant interaction between the two factors was found, a simple main effect test was also performed. In Fig. 5A, the statistical significances between 2 groups were examined by the unpaired t-test. In Figs. 5B, 6B,C for multiple comparisons, we performed 1-way ANOVA, followed by the SNK test. A difference with p < 0.05 was considered to be statistically significant. www.nature.com/scientificreports/

Data availability
The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.